Pre-eclampsia is a syndrome of hypertension, edema, and proteinuria that affects 5 to 10% of pregnancies and results in substantial maternal and fetal morbidity and mortality. Pre-eclampsia accounts for at least 200,000 maternal deaths worldwide per year. The symptoms of pre-eclampsia typically appear after the 20th week of pregnancy.
Development of a fetus and placenta is mediated by several growth factors. Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen, and angiogenic inducer. VEGF mediates vascular permeability and has been shown to be involved in glomerular capillary repair. VEGF binds as a homodimer to one of two homologous membrane-spanning receptor tyrosine kinases, the fms-like tyrosine kinase (Flt-1) and the kinase domain receptor (KDR).
Placental growth factor (PlGF) is a VEGF family member that is also involved in placental development. PlGF is expressed by cytotrophoblasts and syncytiotrophoblasts and is capable of inducing proliferation, migration, and activation of endothelial cells. PlGF binds as a homodimer to the Flt-1 receptor, but not the KDR receptor. Both PlGF and VEGF contribute to the mitogenic activity and angiogenesis that are critical for the developing placenta.
A soluble form of the Flt-1 receptor (sFlt-1) has been identified in a cultured medium of human umbilical vein endothelial cells and in vivo expression was subsequently demonstrated in placental tissue. sFlt-1 is a splice variant of the Flt-1 receptor which lacks the transmembrane and cytoplasmic domains. sFlt-1 binds to VEGF with high affinity but does not stimulate mitogenesis of endothelial cells. sFlt-1 is believed to act as a “physiologic sink” to down-regulate VEGF signaling pathways. Regulation of sFlt-1 levels therefore works to modulate VEGF and VEGF signaling pathways. Careful regulation of VEGF and PlGF signaling pathways is critical for maintaining appropriate proliferation, migration, and angiogenesis by trophoblast cells in the developing placenta.
A single gene codes for human PlGF. However, splicing of the mature PlGF mRNA results in three different length isoforms: PlGF-1 (PlGF131), PlGF-2 (PlGF152), and PlGF-3 (PlGF203). Another variant, PlGF-4, has been reported (Yang, et al, J Reprod Immunol, v 60, p 53-60, 2003). PlGF is secreted as a glycosylated homodimer.
Recently it has been shown that sFlt-1 and PlGF may be used individually or in combination as biomarkers to predict, diagnose, or monitor pre-eclampsia (Levine et al, NEJM, v 350, p 672-683, 2004).
The amino acid sequence of mature human PlGF-1, amino acid residues 1-132, has been published and is available from the Protein Data Bank identified as PDB 1FZV (Iyer, et al, J. Biol Chem, v 276, p 12153-12161, 2001). This sequence is identified herein as SEQ ID NO:1:
MLPAVPPQQW ALSAGNGSSE VEVVPFQEVW GRSYCRALER LVDVVSEYPS EVEHMFSPSC VSLLRCTGCC GDENLHCVPV ETANVTMQLL KIRSGDRPSY VELTFSQHVR CECRPLREKM KPERCGDAVP RR
Diagnosis of an individual at risk for, or having pre-eclampsia may be made by determining the presence or amount of vascular endothelial growth factor, particularly PlGF, and/or receptor tyrosine kinase, particularly, sFlt-1 in a biological sample (such as urine, whole blood, serum, plasma, saliva, and so forth) taken from the individual. In analytical assays reference, calibrator and control compositions are essential for purposes of determining the amount or confirming the presence of a target analyte and, for establishing accuracy and precision of the analytical assay. The preparation of such compositions in liquid or dry form usually doesn't present difficulties if the analyte is readily available, soluble in an appropriate solvent—usually aqueous for biological analytes, stable, and does not interact deleteriously with other components that may be present in the composition. As noted above, PlGF binds sFlt-1 to form a stable association complex. As a result compositions comprising the native proteins together in independent amounts suitable for use as a reference, calibrator or control in analytical assays to detect PlGF or sFlt-1 or both PlGF and sFlt-1 cannot be prepared. Although compositions comprising the individual, separated proteins may be prepared it would be advantageous to be able to prepare compositions comprising both proteins together. Thus, a need exists for reference, calibrator or control compositions comprising these proteins together in known and independent amounts. This need has been met with the present invention.